2-methoxyethyl 2-cyanoacrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

HET-CAM Test

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study report data

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: HET-CAM Test

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures

Principles of method if other than guideline:

With the HET-CAM the irritation property of a test item after contact with mucous membranes or the eye can be determined based on the occurrence of alterations at the CAM and the adjacent capillary system of fertilized and incubated chicken eggs.
The chorioallantoic membrane (CAM) is a vital vascular membrane with a closed blood vessel system. After application of a test item on the membrane the underlying blood vessels could become damaged. In particular three events are observed:
- hemorrhage (H) (bleeding out from the vessels of the vascularized CAM)
- vessel lysis (L) (disappearance of small vessels)
- protein coagulation (C) (intravasal or extravasal)
Two different methods can be used depending on the transparency of the test item, the reaction-time method or the end-point assessment. Since 2-methoxyethyl 2-cyanoacrylate was shown to polymerize and harden on the CAM, the end-point assessment was applied.

The study procedures were based on the following document:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Hen’s Egg Test - Chorioallantoic Membrane (HET-CAM) test method, March 2006.
- Steiling et al. (1999) The HET-CAM, a useful in vitro assay for assessing the eye irritation properties of cosmetic formulations and ingredients. Toxicology in Vitro 13, 375-384 (1)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxyethyl 2-cyanoacrylate

EC Number:

248-670-5

EC Name:

2-methoxyethyl 2-cyanoacrylate

Cas Number:

27816-23-5

Molecular formula:

C7H9NO3

IUPAC Name:

2-methoxyethyl 2-cyanoprop-2-enoate

Test material form:

liquid

Details on test material:

2-methoxyethyl 2-cyanoacrylate
Test Item name: SAT 130032
Batch: #L2PW120143

Test animals / tissue source

Species:

other: HET-CAM

Strain:

other: not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

Amount / concentration applied:

300 µL were topically applied to the CAM of 6 individual eggs each.

Duration of treatment / exposure:

300 s

Number of animals or in vitro replicates:

6

Details on study design:

On day 10, after nine days incubation, the eggs were candled to check the correct development of the fertilized egg as well as to visualise air chamber position of individual eggs. Along the marked air chamber the eggshell was carefully sawed and the CAM was uncovered. Only eggs with a marked blood vessel system were used for the test. Eggs with a non-marked vessel system were discarded.

300 μL of the test item and the same amount of Texapon ASV Spezial (5 %) as a reference were topically applied to the CAM of 6 individual eggs each. The eggs were exposed for 300 seconds to the test item and the reference compound. Results for the three test method endpoints were evaluated separately for each egg and the S-score was calculated. The test item was classified by means of the strongest reaction type.

Classification scheme of the HET-CAM End-Point Assessement:
S [sum of single value] Evaluation
0 – 5 slightly irritating
6 – 12 moderately irritating
13 – 15 irritating
16 – 18 severely irritating (R 41)

Results and discussion

In vitro

Results

Irritation parameter:

other: HET-CAM S-score

Run / experiment:

mean 300 s treatment

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

	Egg No.	Severity Score of Haemorrhage	Severity Score of Lysis	Severity Score of Coagulation
Test Item	1	1*	0	0
	2	1	0	0
	3	1	0	0
	4	1*	0	0
	5	2*	0	0
	6	2	0	0

*When lifting and removing of the test item from the CAM, a vessel was injured (mechanical damage)

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: ICCVAM

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses a moderate irritating potential to mucous membranes or eyes.

Executive summary:

The irritation potential of 2 -methoxyethyl 2 -cyanoacrylate to eyes and mucous membranes was evaluated in the HET-CAM (Hen's Egg Test – Chorioallantoic Membrane) by analysing specific effects to the membrane and/or vessels of the CAM of 9 day incubated chicken eggs. The specific sensitivity of the batch of eggs could be shown by means of the reference item.

2 -Methoxyethyl 2 -cyanoacrylate was tested undiluted. After application, the test item started to harden within 30 seconds on the CAM and became turbid. Since the CAM became invisible due to the test item's hardening process effects were evalutated using the endpoint method (EPM). After completion of the first 30 seconds of exposure of test item to the CAM according to the EPM, the test item could not be rinsed off. Therefore, the exposure period was prolonged to 5 minutes to have the hardening process completed. Afterwards, the hardened test item could be carefully lifted and removed as one piece.

The occurrence of reactions on the blood vessels under the CAM was monitored after removing of the test item.

The individual test results were interpreted in comparison to the effects of the reference item Texapon ASV Spezial which is defined to be a moderately irritating compound to the rabbit eye in vivo. The dilutions of Texapon ASV Spezial with 0.5%, 1%, and 5% active substance (AS) in deionised water were used as reference for the scoring of the effects on the CAM caused by the test item. After 5 minutes of treatment with Texapon ASV Spezial dilutions, the 0.5% solution demonstrated weak effects, the 1% solution moderate effects, and the 5% solution strong effects on the CAM.

Coagulation or lysis were not observed after exposure of the test item to the CAM, but slight to moderate haemorrhaging effects were visible. However, in three of six eggs lifting resulted in mechanical damage of vessels. By also considering those results where mechanical damage occured, the highest summed up score was 8 for haemorrhage. Consequently, the test item is classified as moderately irritating to mucous membranes or eyes.